Antibiotic antimycotic

To isolate hUCB-MNCs, 50 mL hUCB was procured ex utero from each subject immediately after baby delivery. MNCs were isolated from the hUCB sample
using a Lymphoprep medium (StemCell Technologies, Canada). Subsequently, the isolated hUCB-MNCs were then cultured in MesenCult media
with a stimulatory supplement (StemCell Technologies, Canada) containing antibiotic-antimycotic (Sigma-Aldrich, USA) in a humidified
incubator (5% CO₂, 37 °C).

To directly identify a functional role for K_ir2.1 channels, the coronary arteriolar dilation in response to K⁺ was examined in the presence of antisense or sense oligonucleotides according to the Superfect Transfection Reagent protocol (Qiagen, Germantown, MD, USA) [60 (link),61 (link)]. Coronary arterioles (60–100 µm in diameter in situ) were carefully dissected and incubated in Dulbecco’s Modified Eagles Medium containing 10% fetal bovine serum, 1% antibiotic–antimycotic (Sigma), and Superfect Transfection Reagent (20 µL/mL) for 3 h at 37 °C in the absence and presence of antisense or sense oligonucleotides (2.5 µg/mL) for K_ir2.1 channels (22 base pairs corresponding to bases 381–402 of gene accession no. AF153820, synthesized by Sigma-Genosys) [62 (link)]. After this incubation period with the transfection–oligonucleotide complexes, the media was removed, and the vessels were then incubated with the media alone for 24 h. The vessels were subsequently cannulated and pressurized for functional assessment in response to K⁺. RT-PCR analysis was performed as described below to verify the efficacy of K_ir2.1 blockade by antisense oligonucleotides.

A mixture of 0.5 mL heparinized blood and 4.5 mL RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 2 mM L-glutamine (Sigma, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, São Paulo, Brazil), 100 μg/mL antibiotic-antimycotic (Sigma, St. Louis, MO, USA) and 2% phytohemagglutinin (Sigma, St. Louis, MO, USA) was prepared. Aliquots were incubated at 37 °C for 44 h under 5% CO₂. After incubation, 6 μg/mL cytochalasin B (Sigma) was added to inhibit cytokinesis. Lymphocytes were then collected by centrifugation at 1200 rpm for 8 min, fixed in methanol/acetic acid solution (25:1, v/v), mounted on clean slides and stained with Diff-Quik (Medion Diagnostics, Düdingen, Switzerland). To assess DNA damage and chromosome instability indices, we examined 2000 binucleated (BN) cells per blood sample, with 1000 cells evaluated from each duplicate culture slide. Endpoints included micronuclei (MNBN), nucleoplasmic bridges (NPB), and nuclear buds (NBUDs), which were analyzed by light microscopy at 200–1000× magnification [51 (link)]. All slides were coded with unique identifiers to ensure unbiased analysis according to the criteria described by Fenech et al. [52 (link)].

Human keratinocytes were reprogrammed as previously described (Stockmann et al., 2013 (link)) using a lentiviral polycystronic STEMCCA cassette encoding octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4(KLF4), and c-MYC (Somers et al., 2012 (link)). Briefly, keratinocytes (75% confluence) were treated with 5 × 10⁵ proviral genome copies in EpiLife medium (Invitrogen, Carlsbad, CA, USA) supplemented with 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA) on two subsequent days. Afterwards, keratinocytes were detached with TrypLE Express (Invitrogen, Carlsbad, CA, USA) for 10 min at 37°C and transferred to previously irradiated (30Gy) rat embryonic fibroblasts feeder cells. Keratinocytes were cultured in hiPSC medium consisting of knockout/DMEM supplemented with 20% knockout serum replacement, 2 mM GlutaMAX, 100 mM non-essential amino acids (all from Life technologies), 1% Antibiotic–Antimycotic, 100 mM β-mercaptoethanol (Millipore, USA), 50 mg/ml vitamin C, and 10 ng/ml fibroblast growth factor (FGF-2) (both from PeproTech, USA) in a 5% O₂ incubator. Medium was changed daily. After 3–5 days small colonies appeared with typical hiPSCs morphology. Around 14 days later, hiPSC colonies had the appropriate size for mechanical passaging and were transferred onto irradiated MEFs (Stem Cell Technologies, France) and further cultivated with hiPSC medium. After one passage hiPSC colonies were mechanically picked and transferred to feeder free plates and maintained with mTReSR1 medium (Stem Cell Technologies, France). For splitting, hiPSCs colonies were incubated with dispase (StemCell Technologies) for 5–7 min at 37°C and subsequently detached using a cell scraper. For more detailed methods see Stockmann et al. (2013) (link).
The reprogramming of the human fibroblasts was performed essentially as described by Reinhardt et al. (2013) (link) and Lojewski et al. (2014) (link) using pMX-based retroviral vectors encoding human Yamanaka factors (Reinhardt et al., 2013 (link); Lojewski et al., 2014 (link)). For infection, up to 50 000 fibroblasts per well of a 0.1% gelatin-coated 6-well-plate were infected three times with pMX vectors in combination with 6 μg/ml protamine sulfate (Sigma-Aldrich) and 5 ng/ml FGF-2 (Peprotech). Infected fibroblasts were plated onto mitomycin C (MMC, Tocris) inactivated CF-1-MEFs. The next day media was exchanged to ES medium containing 78% Knock-out DMEM, 20% Knock-out serum replacement, 1% non-essential amino acids, 1% penicillin/streptomycin/glutamine and 50 μM β-mercaptoethanol (all from Invitrogen) supplemented with 5 ng/ml FGF-2 and 1 mM valproic acid (Sigma Aldrich). Media was changed every day to the same conditions. Seven days after viral infection hiPSC-like cells appeared and were cultured for additional 7 days. At day 14 post-infection, the cells were manually picked and plated on CF-1 feeder cells in ES medium supplemented with 5 ng/ml FGF-2. By using 1 mg/ml collagenase type IV (Invitrogen) constant colonies were passaged on CF-1 feeder cells (Globalstem, Gaithersburg, MD, USA) treated with MMC. Addition of 10 μM Rock-inhibitor Y-27632 for the first 48 h improved survival of hiPSCs. Medium was changed daily and contained FGF-2. The cultivation of generated hiPSCs at later passages was performed essentially as described (Stockmann et al., 2013 (link)) under feeder- and serum-free conditions.